腺相关病毒转染改良基因ND4进入线粒体.doc

华 中 科 技 大 学 硕 士 学 位 论 文 Adeno-associated virus-mediated modified ND4 gene entering the mitochondria Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Postgraduate: Wan Xing Tutor: Porf. Li Bin Abstract Aim: To investigate AAV-mediated the modified ND4 gene whether could facilitate ND4 protein redirect import to mitochondria. Methods: A nuclear encoded human ND4 gene fused to COX10 mitochondrial targeting sequence (MTS) and COX10 3‘Untranslated Regions (UTR) were packaged in AAV2/2 capsid. Then the recombina HYPERLINK app:ds:respectively nt HYPERLINK app:ds:respectively HYPERLINK app:ds:respectively AAV2/2-MTS-ND4 we constructed were validated by vitro and vivo experimen HYPERLINK app:ds:respectively ts, HYPERLINK app:ds:respectively HYPERLINK app:ds:respectively respectively. HYPERLINK app:ds:respectively In vitro experiment, cells were divided into three groups and were adding PBS, AAV-GFP, rAAV-MTS-ND4 in sequence. The mRNA and protein of ND4 in the Human embryonal kidney cells (293T) were determined with immunofluorescence, Western blot and real-time polymer chain reaction (RT-PCR) after 48 hours. In vivo experiment, animals were divided into three groups and received intravitreal injection 4μl of PBS、AAV-GFP、 rAAV-MTS-ND4 in sequence. The mRNA and protein of ND4 in retina of rabbits were determined with immunofluorescence, Western blot and real-time polymer chain reaction (RT-PCR) at thirtieth days post-injection. Results: Immunofluorescence of cells show ND4 protein were redirect imported to mitochondria of cells. Intravitreal injection of AAV vectors efficiently transduced mainly retinal ganglion cells. Western blot analyses verified the significantly increased expressions of ND4 protein into mitochondrial of 293 cells and retinal ganglion cells of mouse in group 3 than the other two groups. Real-time polymer chain reaction (RT-PCR) detection verified the significantly increased mRNA of ND4